METALLOPROTEIN STRUCTURES WITH THREE FOLDS

该提议的总体目标是提高对金属蛋白中生物学上重要的过渡金属离子的结合和活性的结构基础的理解，通过工程化、表征和确定掺入移植的金属结合位点的定点突变蛋白的X射线结构来测试这种理解，并产生模拟或扩展原始金属结合位点的金属结合和催化性质的设计的金属蛋白。我们将使用基于结构的递归设计，从已知结构的含Cu和Zn的金属蛋白中移植金属离子模板到三种不同的蛋白质结构框架中。对于蛋白质支架，我们将从抗体、光敏黄蛋白（PYP）和绿色荧光蛋白（GFP）的已知高分辨率晶体结构开始。通过使用相同的金属离子模板与不同的蛋白质支架和在一个单一的支架内的不同位置，我们将测试蛋白质框架的作用，在确定金属位点的几何形状，并提供所需的亲和性，特异性和活性的蛋白质中的金属结合位点的环境。在设计和构建，生化和光谱表征，和三维结构的测定和分析这些金属蛋白突变体后，我们将应用我们的结果和其他项目的结果来改进和优化原始设计，或建议新的设计。从其他项目的结果在折叠和组装，稳定性，调节和金属蛋白的化学活性的金属位点的结构和化学作用将有助于实验和计算信息，以改善我们的金属蛋白设计。这种互动的方法将使我们能够严格测试和完善我们对蛋白质中功能金属位点的要求的理解。该提案的长期目标是设计科学和医学上有用的金属蛋白，其协同组合抗体的特异性或PYP和GRP的天然报告基团，与蛋白质结合的金属离子的显著功能和催化性质。

The overall goals of this proposal are to improve understanding of the structural basis for the binding and activity of biologically important transition metal ions in metalloproteins, to test this understanding by engineering, characterizing, and determining x-ray structures for site- directed mutant proteins that incorporate transplanted metal-binding sites, and to produce designed metalloproteins that mimic or extend the metal-binding and catalytic properties of the original metal-binding site. We will use recursive structure-based design to transplant metal- ions templates from Cu- and Zn-containing metalloproteins of known structure into three different protein structural frameworks. For protein scaffolds, we will start from the known, high resolution, crystallographic structures of antibodies, photoactive yellow protein (PYP) and green fluorescent protein (GFP). By using the same metal ion templates with different protein scaffolds and among different locations within a single scaffold, we will test the roles of the protein framework in determining metal-site geometry and providing the environment needed for the affinity, specificity, and activity of metal- binding sites in proteins. After design and construction, biochemical and spectroscopic characterization, and 3-dimensional structure determination and analysis of these metalloprotein mutants, we will apply our results and those from other projects to improve and optimize the original designs, or to suggest new designs. The results from the other projects on the structural and chemical roles of metal sites in the folding and assembly, stability, regulation, and chemical activity of metalloproteins will contribute experimental and computational information to improve our metalloprotein designs. This interactive approach will allow us to rigorously test and refine our understanding of the requirements for functional metal sites in proteins. The long term objective of this proposal is to engineer scientifically and medically useful metalloproteins that synergistically combined the specificity of antibodies or the natural reporter groups of PYP and GRP, with the remarkable functional and catalytic properties of protein-bound metal ions.